A troponin-t mutation initiates cardiomyopathy due to impaired contractile inhibition in Drosophila melanogaster. Anthony Cammarato¹, Meera Cozhimuttam Viswanathan¹, Gaurav Kaushik², Adam J. Engler², William Lehman³. 1) Johns Hopkins University, Baltimore, MD; 2) University of California, San Diego, San Diego, CA; 3) Boston University School of Medicine, Boston, MA.

   Muscle contraction results from a series of orchestrated molecular events that involve transient interactions between myosin-containing thick and actin-containing thin filaments. Regulation of striated muscle contraction is primarily achieved by Ca²⁺-dependent modulation of myosin crossbridge cycling on actin by the thin filament (TF) troponin-tropomyosin complex. Alterations in various subunits of the complex trigger contractile dysregulation and myopathy. For example, point mutations located over a span of ten amino acids (130-39) of human cardiac troponin T (cTnT) are associated with distinct cardiomyopathic responses. The Drosophila up¹⁰¹ (E88K) mutation localizes to the end of this well-conserved region of TnT. Here, using multiple image-based approaches we define the consequences of the lesion on the fly cardiac tube. Direct immersion DIC optics, high-speed video imaging and motion analysis resolved a phenotype reminiscent of human restrictive cardiomyopathy in up¹⁰¹ hearts. Relative to controls, end-diastolic and end-systolic dimensions and percent fractional shortening were significantly reduced. Furthermore systolic intervals were significantly prolonged. This suggests TF dysregulation initiates excessive periods of force production and diastolic dysfunction. Electron microscopy and three-dimensional reconstruction of TFs revealed the vast majority of Ca⁺²-free mutant TFs exhibited tropomyosin in a position distal to known myosin binding sites where it is unlikely to prevent crossbridge formation. Finally, atomic force microscopy and nanoindentation identified elevated up¹⁰¹ cardiomyoctye stiffness in the absence of Ca²⁺ that was attenuated via incubation with a myosin-specific inhibitor. This is consistent with unregulated active forces contributing to incomplete relaxation. Thus, as found in humans with distinct cTnT mutations, up¹⁰¹ TnT likely promotes TF dysinhibition and consequently restrictive cardiac remodeling.